Carotenoids are widely distributed over green plants. They are yellow-orange-red lipids which are also present in some mold, yeast and so forth, and have recently received increased attention as natural coloring materials for foods. Among these carotenoids, .beta.-carotene is a typical one, which is used as a coloring materials and as a precursor of vitamin A in mammals as well. It is also expected to be useful for preventing cancer [see, for example, SHOKUHIN TO KAIHATSU (Foods and Development), 24, 61-65 (1989)]. Since carotenoids such as .beta.-carotene are widely distributed over green plants, plant tissue culture has been examined as a means to produce carotenoids in a large amount without the influence of natural environment [see, for example, Plant Cell Physiol., 12, 525-531 (1971)]. Examination has also been made to search for a microorganism such as mold, yeast or green algae with a high carotenoid producing activity and to produce carotenoids in a large amount using such microorganism (see, for example, The Abstract of Reports in the Annual Meeting of NIPPON HAKKO KOGAKU-KAI of 1988, page 139). However, neither of these is successful at present in producing .beta.-carotene at a good productivity which exceeds synthetic methods in commercial production. It would be very useful to obtain a gene group which participates in the biosynthesis of carotenoids, because it will be possible to produce carotenoids in a larger amount by introducing and expressing appropriate gene(s) among such group in an appropriate host such as a plant tissue cultured cell, a mold, a yeast or the like which originally produces carotenoids. This will lead to a .beta.-carotene production superior to synthetic methods, or to the production of useful carotenoids other than .beta.-carotene in a large amount.
Furthermore, the gene group participating in the biosynthesis of carotenoids would make it possible to synthesize carotenoids in a cell or an organ which originally has no carotenoid producing ability, which will add new values to the organism. For example, several reports have recently been made with reference to creating flower colors not found in nature by the genetic manipulation of flowering plants [see, for example, Nature, 330, 677-678 (1987)]. Flower colors are given by pigments such as anthocyanin and carotenoids. Anthocyanin is responsible for flower colors of red-violet-blue, and carotenoids are responsible for flower colors of yellow-orange-red. Genes encoding enzymes involved in anthocyanin synthesis have been elucidated, which were used for creating a new flower color in the aforementioned reports. On the other hand, there are many flowering plants having no bright yellow flower due to lack of carotenoid synthesis in petals (e.g. petunia, saintpaulia (African violet), cyclamen, Primula malacoides, etc.). Expression of appropriate reconstructed gene(s) for carotenoid biosynthesis in petals would give yellow flowers to these flowering plants.
However, enzymes for synthesizing carotenoids or genes coding for them are scarcely elucidated at present. The nucleotide sequence of the gene group participating in the biosynthesis of a kind of carotenoids has been elucidated lately in a photosynthetic bacterium Rhodobacter capsulatus [Mol. Gen. Genet., 216, 254-268 (1989)]. This bacterium, however, synthesizes an acyclic xanthophyll spheroidene via neurosporene without cyclization and thus cannot synthesize general carotenoids such as lycopene, .beta.-carotene and zeaxanthin.
There are prior arts with reference to yellow pigments or carotenoids of Erwinia species disclosed in J. Bacteriol., 168, 607-612 (1986), J. Bacteriol., 170, 4675-4680 (1988) and J. Gen. Microbiol., 130, 1623-1631 (1984). The first one of these references discloses the cloning of a gene cluster coding for yellow pigment synthesis from Erwinia herbicola Eho 10 ATCC 39368 as a 12.4 kilobase pairs (kb) fragment. There is no information on the nucleotide sequence of the 12.4 kb fragment. The second literature discloses a yellow pigment synthesized by the cloned gene cluster, which is indicated to be a carotenoid by the analysis of its UV-visible spectrum. The last literature indicates that the genes participating in the production of yellow pigments in Erwinia uredovora 20D3 ATCC 19321 are present on a 260 kb large plasmid from the observation that the yellow pigments are not produced after curing the large plasmid, and further discloses that the pigments are carotenoids flora the analysis of their UV-visible spectrum.
However, the chemical structures of the carotenoids and their precursors produced by the Erwinia species, enzymes participating in their synthesis or the nucleotide sequences of the genes encoding these enzymes have been unknown. Very recently, an international patent application was published (WO 91/13078, published on Sep. 5, 1991) which discloses the isolation and nucleotide sequences of the carotenoid synthesis genes of Erwinia herbicola Eho 10 and their expression in several organisms.